American Society of Hematology

Fig. 3.

$Fig. 3. RPS19 transgene expression in transduced CD34+ cells from patients with DBA. / (A) The retroviral vector MRIG consists of the MSCV retrovirus backbone containing the RPS19 cDNA followed by an IRES from the encepharomyocarditis virus and the GFP gene. To obtain higher transgene expression, we constructed the MND-RIG vector containing the same RPS19-IRES-GFP casette. The MND-RIG vector was derived from the MND-MFG vector which contains the myeloproliferative sarcoma virus LTR without the negative control region and the region between the donor and acceptor splice sites is derived from the MFG vector.24 SD and SA represent the splice donor and splice acceptor sites, respectively. MGIN and MND-GFP are respective control vectors containing the GFP gene without the RPS19 cDNA. (B) Flow cytometric analysis of transduced CD34+ cells. The figure shows a representative experiment demonstrating MRIG and MGIN transduction of CD34+ BM cells from an RPS19-deficient patient with DBA and a healthy individual. The CD34+ cells were exposed to a cocktail of growth factors during the transduction. The CD34+ fractions of the bone marrow cells from a healthy donor and a patient with DBA were transduced with the oncoretroviral vectors MRIG or MGIN. The percentage numbers show the ratios of cells in each of the quadrants. The GFP+ and CD34+cells (box) were sorted by FACS vantage. The controls used to set the gates showed that mock transduced cells were 0.27% CD34+/GFP+ and 0.05% were CD34−/GFP+. (C) Northern blot analysis of the transduced cells. One microgram of total RNA from GFP+ and CD34+ cells were electrophoresed in 1% agarose, transferred onto a nylon membrane, and hybridized with radioactively labeled DNA fragment specific for RPS19. The MRIG GP+envAm12-producing cell line was used as a positive control. The asterisk indicates the endogenous expression of RPS19 (0.5 kb). Arrows indicate the vector generated RPS19 (3.3 kb, unspliced and 2.7 kb, spliced). The mock sample represents mock-transduced CD34+ cells that were not sorted on the FACS.$

RPS19 transgene expression in transduced CD34⁺ cells from patients with DBA.

(A) The retroviral vector MRIG consists of the MSCV retrovirus backbone containing the RPS19 cDNA followed by an IRES from the encepharomyocarditis virus and the GFP gene. To obtain higher transgene expression, we constructed the MND-RIG vector containing the same RPS19-IRES-GFP casette. The MND-RIG vector was derived from the MND-MFG vector which contains the myeloproliferative sarcoma virus LTR without the negative control region and the region between the donor and acceptor splice sites is derived from the MFG vector.24 SD and SA represent the splice donor and splice acceptor sites, respectively. MGIN and MND-GFP are respective control vectors containing the GFP gene without the RPS19 cDNA. (B) Flow cytometric analysis of transduced CD34⁺ cells. The figure shows a representative experiment demonstrating MRIG and MGIN transduction of CD34⁺ BM cells from an RPS19-deficient patient with DBA and a healthy individual. The CD34⁺ cells were exposed to a cocktail of growth factors during the transduction. The CD34⁺ fractions of the bone marrow cells from a healthy donor and a patient with DBA were transduced with the oncoretroviral vectors MRIG or MGIN. The percentage numbers show the ratios of cells in each of the quadrants. The GFP⁺ and CD34⁺cells (box) were sorted by FACS vantage. The controls used to set the gates showed that mock transduced cells were 0.27% CD34⁺/GFP⁺ and 0.05% were CD34⁻/GFP⁺. (C) Northern blot analysis of the transduced cells. One microgram of total RNA from GFP⁺ and CD34⁺ cells were electrophoresed in 1% agarose, transferred onto a nylon membrane, and hybridized with radioactively labeled DNA fragment specific for RPS19. The MRIG GP⁺envAm12-producing cell line was used as a positive control. The asterisk indicates the endogenous expression of RPS19 (0.5 kb). Arrows indicate the vector generated RPS19 (3.3 kb, unspliced and 2.7 kb, spliced). The mock sample represents mock-transduced CD34⁺ cells that were not sorted on the FACS.

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